G protein-coupled receptors are a family of cell surface receptors that regulate G protein signaling pathways in response to a variety of environmental stimuli. These events are important in the control of growth, differentiation and metabolism in eukaryotic cells through the stimulation of second messengers, phosphorylation cascades and the regulation of ion channels. Control of the lifetime of an active receptor through a process known as desensitization is a critical aspect of the regulation of G protein signaling pathways. Rhodopsin, the photoreceptor of the vertebrate rod cell, has been used as a structural model for investigating the interactions between G protein-coupled receptors and their G proteins, as well as the G protein-coupled receptor kinases and arrestins that mediate receptor desensitization. Previously, our laboratory identified several nonoverlapping domains on the surface of rhodopsin that are involved in Gt activation, interaction with rhodopsin kinase and arrestin. The aim of the present proposal is to provide an in-depth understanding of the molecular roles played by domains that we have identified in the regulation of receptor desensitization. In order to achieve this goal, clustered alanine mutations will be separated into individual alanine point mutants and tested for their ability to be phosphorylated and to bind arrestin. Additional mutations will be made at these sites to determine the requirement for charge, hydrophobicity, size or specific secondary structure. The mutants that affect phosphorylation will be analyzed to determine whether these sites are important for binding rhodopsin kinase, regulating its activity, or both. Mutants that affect arrestin binding will be examined to determine whether they are part of a high- affinity binding site on the surface of rhodopsin or whether they are important in regulating the transition of arrestin from a low to a high affinity form. Mutants of the highly conserved amino acid Arg-135, was shown by our laboratory to be phosphorylated and to bind arrestin in the absence of 11-cis-retinal. Additional substitution mutagenesis will be performed to analyze the requirement for specific amino acids or secondary structure at this site. The consequences of retinal- independent phosphorylation and arrestin binding on the ability of these mutants to activate Gt will also be performed. A better understanding of the mechanisms by which the cytoplasmic domains of rhodopsin control G protein activation and desensitization will aid in defining potential mechanisms of disease resulting from defects in G protein-coupled receptor signaling pathways and in designing therapeutic strategies.